Bacterial DNA replication is carefully controlled at the initiation stage, possibly by the regulation of the essential activity of DnaA protein activity. The cellular membrane has long been hypothesized to be involved in chromosomal replication, with accumulating evidence that indicates membranes have a profound influence on DnaA protein. The long term goal of this research is to elucidate the physiological significance of the influence of membranes on chromosomal replication. The research outlined here uses both physiological studies and biochemical approaches with defined components to directly test the hypothesis that membranes participate in the regulation of DnaA protein activity. The specific aims are to: 1. Identify the membrane binding site on DnaA protein by chemical crosslinking. Disrupt the membrane binding function by site- directed mutagenesis and isolate the constructed mutant DnaA proteins. Confirm with defined components that the other replication activities of DnaA protein have not been altered. Examine in vivo the replication activity of DnaA proteins defective specifically for phospholipid binding. 2. Localize DnaA protein within the cell by sub-cellular fractionation and electron microscopic immuno-thin sectioning. Determine if a flux in the cellular location of DnaA protein between the membrane and the cytosol occurs during the cell cycle and with different growth conditions, and if such a change is a means of regulating DnaA protein activity. 3. Examine, with defined components, how being associated with membranes affects the known initiation of replication activities of DnaA protein. This work should provide insight into the regulation of the initiation of DNA replication, which is a key control point in the prokaryotic cell cycle and in the determination of eukaryotic cellular quiescence or proliferation.